Oral fluid rapid assay for hepatitis C virus (HCV) antibodies using non-antibody labeling of IgA molecules recognizing HCV peptide epitopes

ABSTRACT

A method and device to detect Hepatitis C (HCV) antibodies in oral fluid is provided. This method introduces a non-antibody detection molecule that labels all classes of patient antibodies in oral fluid, followed by the specific concentration of labeled anti-HCV antibodies by selective capture in a trapping zone consisting of peptide antigens derived from the HCV genome. Signal generated by the labeled antibodies present in the trapping zone is proportional to the number of anti-HCV antibodies bound to the antigens present in the trapping zone. Presence of signal derived from the capture of antibody/detection molecule complexes in the trapping zone is indicative of past exposure to HCV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/912,405, filed Aug. 5, 2004, which was a continuation-in-part of U.S.patent application Ser. No. 09/938,131, filed on Aug. 23, 2001, whichclaimed the benefit of U.S. Provisional Patent Application Ser. No.60/227,254, filed Aug. 23, 2000.

FIELD OF THE INVENTION

The ability to detect anti-HCV in oral fluid is useful for the rapiddetection of HCV exposure by non-invasive means. The methods provided inthe invention are also useful in the early detection of HCV infection byrecognition of anti-HCV of the IgA class, monitoring of antiviraltherapy, genotyping of HCV virus, determining immune response toindividual HCV epitopes, and monitoring potential vaccination programs.

BACKGROUND OF THE INVENTION

Hepatitis C(HCV) is the major cause of parenterally transmitted non-A,non-B hepatitis (Choo et al., 1989 Science 244:359-362; Kuo et al.,1989, Science 244:362-364) with a prevalence of 1-3% throughout theworld (Davis et al., 1998, Hepatology 28(Suppl 4, pt 2):99A). Chronicdisease develops in 60-85% of patients, with cirrhosis representing amajor hallmark of HCV infection. Among patients whose infectionprogresses to cirrhosis, as many as 1-4% develop hepatocellularcarcinomas annually (Fattovich et al., 1997, Gastroenterology112:463-472). It is estimated that the need for hepatic transplantationfor infected individuals will increase 5-7 fold in the next 20 yearsunless more effective treatments and preventative programs areintroduced (Davis et al., 1998, Hepatology 28 (Suppl 4, pt 2):99A).

While additional anti-viral therapies are needed to combat the spread ofHCV, equally necessary is the development of a rapid, highly sensitiveand cost-effective test to detect and monitor HCV within the population.Current PCR and ELISA-based assays for the detection of HCV are costly,relatively slow and reliant upon serum or plasma as the sample fluid.The substitution of oral fluid for serum in HCV assays would provide acost-effective, non-invasive means to conduct routine screening andwould facilitate sample procurement from patient groups where serumcollection is difficult, such as intravenous drug users, who constitutea significant portion of total HCV cases.

A number of oral fluid-based assays have been designed for the detectionof viral antibodies with good results. Virus-specific antibodies havebeen detected in the oral fluid of patients infected with humanimmunodeficiency virus (Major et al., 1991, J. Infect. Dis.163:699-702), hepatitis A (Stuart et al., 1992, Epdiem. Infect.109:161-166), hepatitis B (Ben Aryeh et al., 1985, Arch. Oral Biol.30:97-99), rubella (Saleh, 1991, J. Egypt Public Health Assoc.66:123-124) and following immunization against polio (Zaman et al.,1991, Acta Paediatrica Scan. 80: 1166-1173), rotavirus (Ward et al.,1992, J. Med. Virol. 36: 222-225) and hepatitis A (Laufer et al., 1995,Clin. Infect. Dis. 20:868-871). For HCV, ELISA-based assays developedinitially for use with serum or plasma have been modified to detectanti-HCV antibodies in oral fluid (Cameron et al., 1999, J. ViralHepatitis 6:141-144; Elsana et al., 1998, J. Med. Virol 55:24-27;McIntyre et al., 1996, Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884;Sherman et al., 1994, Amer. J. Gastroent 89:2025-2027; Thieme et al.,1992, J. Clin. Microbiol. 30:1076-1079); using a modified protocol withthe HCV 3.0 ELISA (Ortho Diagnostic Systems), (McIntyre et al. 1996,Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884) detected anti-HCVantibodies within a group of 18 HCV(+) and 49 HCV(−) oral fluid sampleswith 72% sensitivity and 98% specificity. In the same study, 100%sensitivity and 100% specificity was achieved using the Monolisa HCVassay (Sanofi Pasteur Diagnostics, France). It is unclear what thedifferences were that lead to the increased sensitivity of the Monolisatest, and thus care must be taken in the interpretation of resultsobtained from tests not designed specifically for use with oral fluid.None of these assays has achieved the sensitivity required for a rapidpoint of care test. None of these assays has disclosed the special roleof oral fluid IgA in human oral fluid as a key determinant ofsensitivity and specificity for HCV screening.

An intrinsic difficulty in designing oral fluid-based diagnostic assays,however, is detecting a sufficient proportion of the relatively lowlevels of antibody present in oral fluid to generate a meaningfuldiagnostic result. Indeed, it is estimated that overall antibody levelsare 800-1000-fold lower in oral fluid than in serum (Parry et al., 1987,Lancet 2:72-75) making detection sensitivity of the utmost importance inoral fluid-based tests. While this problem is significant, an HCV assaydesigned to be used specifically with oral fluid as the diagnosticfluid, and not simply a serum-based assay modified for use with saliva,could overcome this complication and provide an important test for HCVin the population.

SUMMARY OF THE INVENTION

The invention disclosed is a means to detect antibodies against HCVusing oral fluid as a sample medium. Assays in the prior art have notachieved the sensitivity and specificity required to rapidly screen HCVinfection in human oral fluid. Most critically, the use of a labeleddetection molecule that recognizes not only IgG, but all classes ofimmunoglobulins, enhances the ability to detect anti-HCV in oral fluidin an ELISA format or using a flow-through system. When detectinganti-HCV using a labeled detection molecule that recognizes onlyanti-HCV of the IgG class, detection sensitivity was vastly reduced. Theincorporation of a detection method that labels multiple classes ofanti-HCV, on the other hand, allows for increased detection sensitivityof samples that would otherwise be scored negative using a detectionmethod that only recognizes IgG.

By coupling this detection method to an assay that utilizes a membranewith immobilized HCV peptide antigens present as a trapping zone,followed by subsequent flow of sample through the trapping zones andselective binding of labeled antibodies specific for HCV epitopes withinthe trapping zone, an immunoassay for the detection of anti-HCV can beperformed in a short time period (<15 minutes). The ability to use oralfluid as a sample is of great value to such a rapid diagnostic toolsince oral fluid can be collected rapidly and used immediately followingcollection. An assay using oral fluid, performed on a miniature testplatform, analyzed in a small light gathering machine, and able to becompleted within 15 minutes from start to finish would be of enormousvalue as a screening agent for HCV in the population. By decreasing thetime of the assay and eliminating the need for invasive blood-basedsample acquisition, such an assay would certainly increase the abilityto screen, detect and monitor HCV within the population.

The use of an assay to detect anti-HCV in saliva would also be ofbenefit in the rapid and non-invasive detection of antibodies followingvaccinations and monitoring of vaccination efficacy over time,monitoring therapeutic response of patients to treatment regimes andscreening for early infection, as IgA antibodies are known to be animportant part of the early stages of the immune response.

Thus, the present invention seeks to overcome the deficiencies of theprior technology by designing an HCV assay that would meet the followingobjectives. A first objective is that the test is non-invasive,generates minimal risk of infection to those administering the test andcan be performed from start to finish by non-medical personnel.

A second objective is that the test is rapid (<15 min.).

A third objective is that the test is specialized to detect the specificclass of anti-HCV antibodies in oral fluid, and is not simply amodification of a current serum-based assay.

A fourth objective is that the test incorporates a number of differentHCV antigens to minimize false negative results.

A fifth objective is that the test is adaptable to future incarnationsof the assay to meet specific diagnostic needs, and that it is sensitiveenough to detect extremely low levels of anti-HCV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Characterization of multiple classes of anti-HCV present inserum and oral fluid. Paired serum/oral fluid samples were screened byHCV 3.0 ELISA using enzyme-conjugated antibodies specific for human IgG,IgM or IgA, respectively. (A) In serum, high levels of anti-HCV IgG andIgM class antibodies are detectable, while relatively little anti-HCVIgA is present. (B) In oral fluid, the majority of antibodies detectableare of the IgG or IgA class with little or no anti-HCV IgM present.

FIG. 2: Components of the HCV strip immunoassay. (A) Top view ofdisassembled assay cassette showing the position of the nitrocellulosetest strip as well as the top and bottom wicks and the substrate-coatedgelbond. The “trapping zone” is located directly beneath thesubstrate-coated gelbond. The trapping zone and substrate-coated gelbondare kept from contacting one another until such time as the cassette isinserted into the luminometer for reading. (B) Top view of an assembledcassette with the conjugate hinge in the open position. (C) Top view ofan assembled cassette with the conjugate hinge in the closed position.Also visible in C is the chase injection port and the luminescencemeasuring window. (D) Side view of assembled cassette showing theconjugate hinge in the open position as well as the lever on the back ofthe cassette that is contacted by the Junior luminometer upon insertionto bring the anti-HCV/anti-human-AP complex captured in the trappingzone into contact with the substrate-coated gelbond suspended above andthus initiate the luminescence reaction.

FIG. 3: Dose response curve for spiked monoclonal anti-HCV antibodies inan HCV(−) oral fluid sample. Monoclonal anti-HCV antibodies were spikedinto an HCV(−) oral fluid sample to test the ability of the mixedantigen trap to capture anti-HCV antibodies. (A) Dose response curve forspiked monoclonal anti-HCV antibodies present in oral fluid atconcentrations ranging from 0-117 μg/ml. (B) Digital photograph ofnitrocellulose test strips post-stained with NBT/BCIPT corresponding tothe data points in (A). Staining within coherent trapping zones isvisible in all spiked samples while no staining is present in thenon-spiked control.

FIG. 4. Screening of 64 known HCV(+) saliva samples on stripimmunoassay. A cutoff was determined using the mean of 14 HCV(−) salivasamples+2SD. 63/64 known HCV(+) saliva samples generated values abovethe calculated cutoff for a sensitivity of 98.4%.

FIG. 5: Direct visualization of HCV LNSI assays using the NightOwlMolecular Light Imager. To observe the relative amounts of luminescenceproduced by highly immunoreactive oral fluid samples (HCV(++)), weaklyreactive samples (HCV(+)) and HCV(−) samples, luminescence was collected(60 sec exposure time) by the NightOwl Molecular Light Imager. Aluminescence intensity scale is provided for reference with purplerepresenting the most intense regions of luminescence. (A) A highlyimmunoreactive oral fluid sample generated extensive luminescence withinthe collection window just below the closed conjugate hinge assembly.(B) A weakly immunoreactive oral fluid sample. (C) An HCV(−) oral fluidsample.

FIG. 6. Antibody profile of 9 individual patient samples using asix-line peptide trapping zone. Patient samples were passed through sixdifferent antigen trapping zones to observe the immune response profilefor these subjects. Strong responders had high levels of antibodybinding against most of the six peptides while weak responders had lowerlevels of binding within the six trapping zones.

FIG. 7 Diagram of the steps of the process according to one embodimentof the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein represents the ability to detect HCVexposure in oral fluid by labeling and detecting multiple classes ofanti-HCV instead of anti-HCV IgG alone. Saliva is first collected by adevice independent of the test module. A volume of crude saliva is thenadded to the test module wherein it mixes with a detection molecule thatlabels all classes of human antibodies. The antibody-detection moleculecomplex then passes through a trapping zone comprised of immobilized HCVpeptide antigens. Antibody/detection molecule complexes that arerecognize the HCV sequences represented in trapping zone bind and arethus immobilized within the zone (FIG. 7). The addition of a suitablesubstrate for the detection molecule allows for generation of a signalin samples possessing antibodies to HCV and thus correlates with HCVexposure. In a particular embodiment the non-antibody label protein isprotein LA conjugated to an enzyme which generates a chemiluminescentsignal that is read in a luminometer.

EXAMPLES I. Detection of Multiple Classes of Anti-HCV in Oral Fluid toIncrease Detection Sensitivity

The detection of multiple classes of anti-HCV in oral fluid can increasethe detection sensitivity of the Ortho HCV 3.0 ELISA to levelscomparable with those attained using serum samples. Patients for thisstudy were pre-selected from one of eleven participating clinical sitesand shown to be either HCV positive or negative based on a clinicaldiagnosis according to the CDC testing algorithm (Alter et al., 1998).Serum samples were further confirmed by repeat in-house testing usingthe Ortho HCV 3.0 ELISA following the manufacturer's instructions. Oralfluid samples were collected using a Salivette (Sarstedt Research,Germany) whereby a polyester-coated cotton plug is placed in the mouthof the patient until saturation and is then centrifuged in a carriertube for 5 minutes to extract the oral fluid. The Salivette was chosenfor its ease of use and because it does not use a sample buffer todilute the specimens. Paired samples were shipped overnight at 4° C. andprocessed immediately upon arrival. Samples were then stored at −80° C.until testing.

To determine if specific classes of antibodies were preferentiallyenriched in serum or oral fluid samples, the composition of anti-HCVpresent in both fluids was examined. Fourteen paired HCV-positive oralfluid/serum samples (with sufficient volumes of oral fluid for multipleELISA assays) were chosen for ELISA analysis and examined usingsecondary enzyme-conjugated antibodies (Jackson Immunoresearch) thatrecognize only IgG, IgM or IgA, respectively, to identify the differentclasses of anti-HCV detectable in oral fluid (FIG. 1). Modification ofthe HCV 3.0 was necessary to achieve optimal detection sensitivity andspecificity; compared to the manufacturer's instructions for use withserum, oral fluid sample volume was increased from 10 μl to 100 μl perwell and sample incubation time was increased from 1 hour at 37° C. toovernight at 4° C. Furthermore, a more sensitive two-part TMB substratekit (Pierce) was used for all testing in place of the o-phenylenediaminetablets supplied with the HCV 3.0 kit. Analysis of the optical densities(OD) generated by these 14 samples showed that anti-HCV of the IgG andIgM class was most abundant in serum samples (mean OD=1.85, 1.03,respectively), with little IgA class anti-HCV present (OD=0.24; FIG.1A). These samples were not treated for rheumatoid factor, however, andthus it is possible that elevated levels of anti-IgM reactivity in serumsamples may be attributable to the presence of this interferingsubstance (see Genser et al., 2001). In contrast, while IgG (OD=1.10)remained the major class of anti-HCV detectable in oral fluid samplesusing the HCV 3.0 assay, a higher level of anti-HCV IgA (OD=0.42) wasalso detectable, while nearly no anti-HCV IgM was present (OD=0.02; FIG.1B). Statistically, the mean OD of anti-HCV of the IgG and IgM class issignificantly reduced in oral fluid compared to serum (P<0.01), whilethe OD of IgA class anti-HCV is not significantly different (P>0.01).

Unexpectedly, in a number of oral fluid samples possessing low anti-HCVIgG levels, a significant amount of anti-HCV IgA was detectable (FIG.1B) which might contribute to a higher overall OD and thus render apositive result. Indeed, the ability to detect anti-HCV of the IgA classmay also increase the likelihood of detection early on during the courseof infection, as IgA is known to be present during the earliest stagesof the immune responses to infections. (Freihorst and Ogra, 2001).

An investigation was then conducted to determine whether the detectionof multiple classes of anti-HCV antibodies, instead of IgG alone, couldincrease the sensitivity of the Ortho HCV 3.0 ELISA in a modified oralfluid-based format. Paired oral fluid/serum samples from 127 known HCVseropositive and 31 seronegative donors were screened using the HCV 3.0assay according to the manufacturer's instructions using the monoclonalanti-human IgG-peroxidase detection antibody. Using serum samples, 100%sensitivity and specificity with the HCV 3.0 assay was achieved (TableI).

TABLE I Sensitivity and specificity of HCV 3.0 assay using pairedserum/oral fluid samples with different enzyme-conjugated secondaryantibodies. Serum Serum Oral fluid Positive Negative Oral fluid PositiveNegative Positive 103 0 Positive 127 0 Negative 24 31 Negative 0 31Conjugate: Monoclonal anti-human IgG Goat anti-human IgG + IgM + IgASensitivity:  81% 100% Specificity: 100% 100%

Because there is no accepted cutoff value for oral fluid in the HCV 3.0kit, sensitivity and specificity were determined by ROC analysis at the95% confidence interval as well as by determining a cutoff 3.5SD abovethe mean of the 31 HCV negative samples. Using the modified incubationprotocol mentioned previously, along with the anti-IgG conjugateantibody of the HCV 3.0 kit, detection sensitivity was reduced to 81%(103/127) while specificity remained 100%.

Oral fluid samples were then re-screened using a 1:16,000 dilution ofperoxidase-labeled goat anti-human IgG+IgM+IgA antibody cocktail(Kirkgaard and Perry Laboratories, Gaithersburg, Md.) in PBS/1% BSA/10%goat serum instead of the monoclonal anti-human IgG provided with theHCV 3.0 kit. This antibody dilution proved to have the greatestsignal:noise ratio in titration studies and was used in all studies inwhich the antibody cocktail was included. Using this modified protocol,anti-HCV was detected in patient oral fluid samples with 100%sensitivity and specificity by ROC analysis or using the calculated3.5SD cutoff (cutoff=0.026; Table I). All oral fluid samples from HCVpositive individuals that were initially scored negative using the OrthoHCV 3.0 anti-IgG conjugate were subsequently scored positive whendetected using the anti-IgG+IgM+IgA cocktail (Table II).

TABLE II Discrepant analysis of select patient oral fluid samplespossessing low anti-HCV IgG. Oral fluid Serum Conjugate Patient #(anti-IgG) (anti-IgG + M + A) (anti-IgG) 103-19 0.014 >3.5 2.42 109-030.014 2.24 2.25 103-15 0.149 2.60 2.07 109-01 0.213 2.77 2.33 103-010.293 >3.5 2.34 103-37 0.357 2.29 1.56 108-06 0.378 >3.5 2.40

The results indicate that the use of a secondary antibody cocktail thatrecognizes not only IgG, but IgA and IgM as well, may aid in thedetection of the relatively low levels of anti-HCV antibodies presentwithin oral fluid and thus increase detection sensitivity. This increasein detection sensitivity when such an antibody cocktail is used is ingood agreement with data showing that a significant percentage ofanti-HCV antibodies in oral fluid exist in the form of IgA classantibody molecules (see FIG. 1). A recent study by Van Doomum et al.(2001) showed that anti-HCV could be detected with up to 88% sensitivityin oral fluid using a modified protocol with the Mono-Lisa anti-HCV Pluskit. Similar to the Ortho HCV 3.0 assay, however, this kit utilizes ananti-human IgG conjugate antibody, and is therefore incapable ofdetecting IgA class anti-HCV present in oral fluid samples. Furthermore,in contrast to the HCV 3.0 assay, the Mono-Lisa does not incorporateproteins from the core region of the HCV proteome and sensitivity inoral fluid may be reduced by the inability to capture antibodiesdirected against this highly antigenic region. By detecting multipleclasses of antibodies, and through the use of an ELISA with a highpercentage of the total antigenic sequences of HCV coated onto the solidphase, an increase in detection sensitivity to levels comparable tothose obtained from serum-based analysis was achieved.

Thus, the results of this study show that detection of anti-HCV IgG, IgMand IgA in oral fluid samples is essential for correctly diagnosingpatient samples possessing relatively low levels of anti-HCV IgG.Indeed, patient oral fluid samples with low anti-HCV IgG levels willescape detection in immunoassays that recognize only IgG classimmunoglobulins. By effectively increasing the pool of antibodiesdetectable in oral fluid samples it is possible to overcome theintrinsic difficulty of detecting the extremely low levels of antibodiesin oral fluid and allow for the generation of novel non-blood basedimmunoassays.

II. Components of the Test Module for the Oral Fluid Based Lateral FlowImmunoassay

The HCV immunoassay consists of a single nitrocellulose strip with amixture of recombinant HCV antigens immobilized in a trapping zone 2.4cm from the top edge of the strip. The nitrocellulose strip is heldstationary within a custom-made plastic cassette assembly (FIG. 2 A).Oral fluid sample and AP-conjugated goat anti-human IgG+IgM+IgA antibodycocktail are added to the conjugate hinge (FIG. 2 B) creating a complexof anti-HCV bound by anti-human-AP antibodies. Alternatively, Protein LAconjugated to alkaline phosphatase can be used as the detectionmolecule. The hinge is then closed and pressed onto the nitrocellulosetest strip for 5 seconds. 60 μl of chase solution is then added to aport on the top of the cassette located just above the hinge region(FIG. 2 C) facilitating the migration of sample complex down thenitrocellulose test strip toward the trapping zone while simultaneouslywashing unbound conjugate antibody through the trapping zone to thebottom wick to prevent non-specific enzyme luminescence within thetrapping zone. Upon reaching the trapping zone, the anti-HCV antibodypresent in the anti-HCV/anti-human-AP complex binds its cognate antigen,thus ceasing its migration. Dried AP substrate is suspended above thetrapping zone (FIG. 2 A) on a piece of gelbond preventing the substratefrom coming into contact with the anti-HCV/anti-human-AP complex in thetrapping zone until the cassette is inserted into the luminometer. Fourminutes after the addition of the chase solution, the test cassette isinserted into the luminometer. A lever on the back of the cassette isdepressed by the luminometer (FIG. 2 D) bringing the substrate intocontact with the anti-HCV/anti-human-AP complex in the trapping zone,thus initiating the luminescence-generating reaction. Luminescence ismeasured through the window in the top of the cassette for 1 minute.

III. Selection of HCV Peptide Sequences to be Incorporated into TrappingZone of Rapid Immunoassay

Peptide sequences shown to be strongly antigenic were chosen forsynthesis and screening for incorporation into the trapping zone of theHCV immunoassay. U.S. Pat. No. 5,698,390 describes the sequencing of theHCV genome and the use of specific, highly antigenic sequences as toolsfor immunoassay development of blood-based HCV assays. These sequences,however, have not yet been useful in the detection of HCV in oral fluidwith high degrees of sensitivity and specificity for use in screening.Contrary to other assays for HCV exposure, peptides were chosen based onthese sequences instead of recombinant antigens for a number of reasons:firstly, since the nucleotide sequence of HCV is well known and many ofthe strongly antigenic epitopes mapped in detail, highly purifiedpeptides that represent only the strongly antigenic regions of the HCVgenome can be synthesized rapidly and in large quantity at relativelylow cost. Secondly, because new peptide antigens incorporating differentantigenic sequences can be synthesized rapidly, new antigens can beadded, or substituted, for peptides already in the assay with relativeease. Thirdly, by incorporating only the highly antigenic sequences ofthe HCV genome, thus eliminating all non-antigenic sequences, thespecificity of the assay can be improved.

On the basis of data generated by screening 30 patient serum samplesagainst individual peptide antigens in an ELISA format, five HCVsequences were chosen. All 30 serum samples were reactive against atleast one of the five peptide antigens (Table III). The followingsequences represent the amino acids numbers based on Kato et al. (1990)chosen for use: 7-26, 22-41, 1694-1710, 1710-1728 and 1924-1943. Thesesequences represent two peptides from the core region of the HCV genomeand three peptides from the NS4 region, respectively.

TABLE III Selection of peptides for use in the trapping zone. Thirtyserum samples were screened against individual peptides by ELISA. All 30samples showed reactivity against at least one of the 5 peptides tested.Peptide antigen (aa #s) 7-26 22-41 1694-1710 1710-1728 1924-1943Sensitivity (%) 97 97 86 90 83

IV. Rapid Screening of Oral Fluid Samples for Anti-HCV Using an AffinityTrap Immunoassay

Detection of Monoclonal Anti-HCV Antibodies Spiked into Oral Fluid

To determine if low levels of monoclonal anti-HCV antibodies in oralfluid are detectable by the HCV LNSI, a mixture of antibodies againstthe core, NS3, NS4 and NS5 antigens was added to an HCV(−) oral fluidsample for a final concentration of antibody ranging from 0 to 117μg/ml. Since all monoclonals were of the IgG subtype, an AP-conjugatedgoat anti-mouse IgG secondary antibody was used to form theanti-HCV/anti-mouse-AP complex. RLUs for the most concentrated spikedsample were 9-fold higher than those obtained for the 0 μg/ml sample(453584 vs. 50568). From the values obtained a dose response curve wasgenerated with an R²=0.98 (FIG. 3 A).

To observe the binding of the anti-HCV/anti-mouse-AP complex within thetrapping zone visually, cassettes were disassembled followingluminescence measurement and the nitrocellulose strips were then stainedfor 7 min. with NBT/BCIP AP substrate. A coherent trapping zone isvisible for assays employing 1.17 to 117 μg/ml spiked monoclonalantibodies in oral fluid (FIG. 3 B). At 0.12 μg/ml a faint band withinthe trapping zone is present, while at 0 μg/ml no band is visible withinthe trapping zone.

Detection of Anti-HCV in Oral Fluid Samples

Sixty-four known HCV(+) oral fluid samples and 14 known HCV(−) sampleswere screened using the invention. A cutoff value was assigned by takingthe mean values of the 14 HCV(−) samples plus 2 standard deviations.Using this cutoff value, 63/64 of the known patient samples were scoredpositive (FIG. 4) leading to a calculated sensitivity of 98.4%. None ofthe HCV(−) samples obtained values greater than that of the calculatedcutoff for a specificity of 100%.

To visualize the luminescence reaction directly, HCV(+), and HCV(−)saliva samples were imaged by a CCD Light Imager. Assays were conductedin the same manner as described previously with the exception thatsticks were imaged in the NightOwl (Bertold Inc. Germany) to detecttotal luminescence. While HCV(+) samples possess high levels ofluminescence, nearly no luminescence is detectable in the HCV(−) sample(FIG. 4).

V. V. Serial Trapping Zones to Type Antibodies Against Specific Peptidesof the HCV Virus

Six HCV peptide antigens were coated as individual trapping zones onto anitrocellulose matrix to demonstrate the ability of the invention todifferentially display the patient antibody response from individualsubjects. Samples were mixed with Protein LA conjugated to alkalinephosphatase and allowed to migrate through the 6 different trappingzones. During this migration, patient antibodies to the differentantigens were selectively captured in the individual trapping zonesallowing for a more detailed analysis of the patient antibody response.The peptide composition of each trapping zone was as follows: line 1 aa384-403, line 2 aa 7-26, line 3 aa 22-41, line 4 aa 1694-1710, line 5 aa1710-1728, line 6 aa 1924-1943.

Results of this study clearly show that the antibody response ofindividual patients can be dissected using this method. This provides auseful means by which antigens to specific genotypes of the HCV viruscan be coated onto a suitable matrix and patient samples can then bescreened in order to provide information regarding the pattern ofantibody response or the strain of virus present (FIG. 5). Suchinformation is useful in tailoring therapy for individual patients onthe basis of the HCV genotype present.

The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations in so far as they come within thescope of the appended claims or the equivalents thereof.

We claim:
 1. A method for screening for HCV exposure in humans thatutilizes an immunoassay for detection of molecule(s) capable ofrecognizing multiple classes of anti-HCV molecules simultaneously inoral fluid samples comprising the following steps: (a) obtaining asample of oral fluid from a human patient; (b) introduction of alabelled anti-human IgG+IgM+IgA antibody cocktail to label IgG, IgM, andIgA antibodies present in the oral fluid sample; (c) introduction of thelabeled fluid into a flow through affinity matrix having a trapping zonecomprised of immobilized HCV peptide antigens; (d) selectively capturinglabeled antibodies which are specific for the HCV peptide antigenspresent within the trapping zone of the flow through affinity matrix;(e) measuring the binding reaction between the human antibodies and theHCV peptide antigens within the trapping zone, thereby detecting thepresence of or the level of IgG, IgM, and IgA antibodies present in theoral fluid sample, wherein the presence of IgG, IgM, and IgA antibodiesin the oral fluid sample is indicative of HCV exposure in humans to HCVvirus.
 2. The method of claim 1, which uses the generation or absence oflight, and a light-gathering device to measure said light, to measurethe antibody quantity and distribution within the trapping zone.
 3. Themethod of claim 1, wherein the fluid sample is undiluted saliva.
 4. Themethod of claim 1, wherein the labeling molecule is alkalinephosphatase-conjugated goat anti-human IgG+IgM+IgA antibody cocktail. 5.The method of claim 1, wherein the screening is completed in 15 minutesor less.
 6. The method of claim 1, wherein the method achieves 98.4% orhigher sensitivity at a specificity of 100% for a diagnosis of HCVexposure.
 7. The method of claim 1, wherein the time between obtainingthe sample of oral fluid and detecting the level of IgG, IgM, and IgAantibodies present in the oral fluid sample is 15 minutes or less.
 8. Amethod for screening for HCV exposure in humans that utilizes an assayfor detection of molecule(s) capable of recognizing multiple classes ofanti-HCV molecules simultaneously in oral fluid samples comprising thefollowing steps: (a) obtaining a sample of oral fluid from a humanpatient; (b) introduction of a labeling molecule to label IgG, IgM, andIgA antibodies present in the oral fluid sample; (c) introduction of thelabeled fluid into a flow through affinity matrix having a trapping zonecomprised of immobilized HCV peptide antigens; (d) selectively capturinglabeled antibodies which are specific for the HCV peptide antigenspresent within the trapping zone of the flow through affinity matrix;(e) measuring the binding reaction between the human antibodies and theHCV peptide antigens within the trapping zone, thereby detecting thepresence of or the level of IgG, IgM, and IgA antibodies present in theoral fluid sample, wherein the presence of IgG, IgM, and IgA antibodiesin the oral fluid sample is indicative of human exposure to HCVinfection.
 9. The method of claim 8, wherein the labeling molecule is anon-antibody molecule used to tag IgG, IgM, and IgA antibodies with areporter molecule for subsequent detection.
 10. The method of claim 8,wherein the non-antibody molecule is Protein LA.